Project Summary In the post-genomic era, proteomics is the next frontier allowing an in-depth understanding of the function of cellular systems in human diseases and the development of personalized treatments. Unlike the genome, the proteome is dynamic and highly complex due to alternative splicing and post-translational modifications (PTMs). The emerging top-down proteomics is the most powerful method to comprehensively characterize proteoforms that arise from genetic variations and PTMs, which is critical for understanding disease mechanisms and identifying new therapeutic targets. However, data analysis tools for top-down proteomics remain under- developed. With the recent rapid growth of the top-down proteomics community, there is an urgent need to develop a comprehensive analysis platform for top-down proteomics. Herein, we aim to develop MASH Explorer, a comprehensive, universal, and user-friendly software environment for top-down proteomics, which includes both an offline downloadable software package (App) and an online version for data processing and analysis (Portal). The specific aims are: 1) to develop MASH Explorer App, a downloadable software package for top- down proteomics that can process data from various vendor formats and incorporate multiple algorithms for deconvolution and database search with user-friendly graphical interfaces; a novel deconvolution algorithm for high-accuracy data analysis and a novel search algorithm for identification of large proteins will also be developed to address the deficiency in the currently available top-down proteomcis software tools; 2) to develop novel algorithms for the automated identification and quantification of proteoform families with enhanced identification of low-abundance proteoforms; 3) to develop MASH Explorer Portal, a web-based extension of MASH Explorer App for processing and sharing top-down proteomics data. The MASH Explorer Portal features my MASH, a cloud-based personalized space for online data processing, as well as MASH public, a public database for long-term data storage and sharing to facilitate the collaboration among various groups and the entire proteomics community. The successful completion of this project will create a powerful software tool, MASH Explorer, for top-down proteomics. The MASH Explorer software will be freely available to all users. Improving the accessibility of non-proprietary free software solutions will significantly bolster the growth of the top-down proteomics community. Hence, we envision it will play integral roles in advancing the burgeoning field of top-down proteomics and realize its full potential for biomedical research.